The present invention generally relates to the provision of nucleic acid from which a polypeptide with utrophin function can be expressed, especially mini-genes and chimaeric constructs. Expression of a utrophin transgene significantly decreases the severity of the dystrophic muscle phenotype in an animal model.
The severe muscle wasting disorders, Duchenne muscular dystrophy (DMD) and the less debilitating Becker muscular dystrophy (BMD) are due to mutations in the dystrophin gene. Dystrophin is a large cytoskeletal protein which in muscle is located at the cytoplasmic surface of the sarcolemma, the neuromuscular junction (NMJ) and myotendinous junction (MTJ). The protein is composed of four domains: an actin-binding domain (shown in vitro to bind actin), a rod domain containing triple helical repeats, a cysteine rich (CR) domain and a carboxy-terminal (CT) domain. The majority of the CRCT binds to a complex of proteins and glycoproteins (called the dystrophin protein complex, DPC) spanning the sarcolemma. This complex consists of cytoskeletal syntrophins and dystrobrevin, transmembrane, xcex2-dystroglycan, xcex1-, xcex2-xcex4-, xcex3-sarcoglycans and extracellular xcex1-dystroglycan. The DPC links to laminin-xcex12 (merosin) in the extracellular matrix and to the actin cytoskeleton via dystrophin within the cell. The breakdown of the integrity of the DPC due to the loss of, or impairment of dystrophin function, leads to muscle degeneration and the DMD phenotype. The structure of dystrophin and protein interactions within the DPC have been recently reviewed [1,2,3].
There are various approaches which can be adopted for the gene therapy of DMD. These include myoblast transfer, retroviral infection, adenoviral infection and direct injection of plasmid DNA. In most cases the dystrophin gene used in the experiments generates a truncated protein approximately half the size of the full size protein. This dystrophin minigene was modelled on a natural mutation identified in a very mild Becker patient [4]. The cloned version of this truncated minigene is able to reverse the pathological phenotype in the dystrophin deficient mdx mouse [5,6,7] and has had limited success when delivered to mdx muscle by viral vectors [8,9,10]. Although some progress is being made in each of these areas using the mdx mouse as a model system, there are problems related to the number of muscle cells that can be made dystrophin positive, the levels of expression of the gene and the duration of expression [11]. Another problem to be addressed is the rejection of cells expressing dystrophin because of immunological intolerance i.e. dystrophin within these cells will appear foreign to the host immune system given that most DMD patients will never have expressed dystrophin [12,13].
In order to circumvent some of these problems, possibilities of compensating for dystrophin loss using a related protein, utrophin, are being explored.
Utrophin is a 395 kDa protein encoded by a gene located on chromosome 6q24 and shown to have strong sequence similarity to dystrophin [14]. The actin binding domain of dystrophin and utrophin has 85% similarity and the DPC binding region has 88% similarity. Both of these domains have been shown to function as predicted in vitro. The structure and potential protein interactions are described in detail in reviews [1,2,3].
There is a substantial body of evidence demonstrating that utrophin is capable of localising to the sarcolemma. During normal fetal muscle development there is increased utrophin expression, localised to the sarcolemma up until 18 weeks and 20 days gestation in human and mouse respectively. After this time the utrophin sarcolemmal staining steadily decreases to the significantly lower adult levels shortly before birth where utrophin is localised almost exclusively to the NMJ and MTJ [15,16,17]. The decrease in utrophin expression coincides with increased expression of dystrophin [17]. Many studies have shown that utrophin is bound to the sarcolemma in DMD and BMD patients. However the levels of utrophin localised at the sarcolemma vary from report to report [18,19,20,21]. In some other non Xp21 myopathies, utrophin and dystrophin are simultaneously bound to the sarcolemma of adult skeletal muscle [22].
High levels of utrophin may protect muscle from the consequences of dystrophin loss. Matsumara et al. [23] demonstrated that purified membranes from the mdx mouse contained complexes of utrophin and the DPC. When quadricep muscles (which show necrosis) from these mice were analysed by immunoblotting, the level of utrophin remained approximately the same, however the level of the xcex1-dystroglycan was drastically reduced. In cardiac muscle (which shows no pathology) the level of utrophin was elevated four fold with no loss of the xcex1-dystroglycan. Immunocytochemical analysis of other mdx small calibre skeletal muscles (extraocular and toe) which also have no pathology shows increased utrophin expression and normal levels of xcex1-sarcoglycan. This result suggests that the increased levels of utrophin interacts with the DPC (or an antigenically related complex) at the sarcolemma and prevents loss of the complex thus the structure of these cells remains normal. In the mdx mouse, utrophin levels in muscle remain elevated soon after birth compared with normal mice; however once the utrophin levels have decreased to the adult levels (about 1 week after birth), the first signs of muscle fibre necrosis are detected [15,16].
Thus, in certain circumstances utrophin can localise to the sarcolemma probably at the same binding sites as dystrophin, namely actin and the DPC. If the expression of utrophin is high enough, it may maintain the DPC and thus alleviate the DMD phenotype. It is unlikely that such external upregulation could be tightly controlled giving rise to potentially high levels of utrophin within the cell. However, this may not be a problem as Cox et al. [24] have demonstrated that gross over expression of dystrophin in the muscle of transgenic mdx mice reverts the muscle pathology to normal with no obvious detrimental side effects.
The present invention has arisen from cloning of nucleic acid encoding utrophin and fragments of utrophin from various species. The original aim was to clone nucleic acid encoding human utrophin, but major problems were encountered. A previous paper (14) reported the amino acid sequence of utrophin (so-called xe2x80x9cdystrophin-related proteinxe2x80x9d), obtained by cloning of overlapping cDNAs. However, two regions around the amino terminal actin binding domain were not represented in these clones. These regions could be amplified by PCR and sequenced, but it has proved not to be possible to clone them. Either clones which should have included these regions were rearranged (as determined by restriction mapping) or simply no clones were isolated even if highly recombination deficient E. coli host strains (SURE and STBL2) were used. The gaps in the sequence were identified by comparing the sequence generated from the utrophin cDNAs to the published human dystrophin sequence. It became apparent as further utrophin clones were isolated, none spanned these two gaps.
Sequence information obtained from the amino terminus of the human cDNA was used to design probes and rat and mouse cDNA libraries were screened. Rat cDNAs were also unstable or rearranged in the region corresponding to the unclonable regions in the human sequence. Some large rat clones covering these regions were obtained, but all attempts to generate subclones failed due to rearrangements of the inserts as determined by restriction mapping. Surprisingly, in view of the difficulties with the human and rat sequences, cDNA from the mouse library, covering the regions in question, was found to be stable and amenable to further manipulation including the. generation of smaller subclones.
FIG. 1 shows a comparison between human, rat and mouse utrophin nucleotide sequences encoding part of the amino-terminal portion of the respective proteins. The unclonable regions of the human gene are underlined.
This cloning work enables for the first time the construction of a nucleic acid molecule from which a polypeptide with utrophin function can be expressed.
Furthermore, by way of analogy with the success achieved with a dystrophin mini-gene (from which a truncated version of dystrophin is expressed) the present invention provides xe2x80x9cutrophin mini-genesxe2x80x9d and polypeptides encoded thereby. To overcome the problem of unclonability of regions of the human utrophin gene sequence, the present inventors have realised that it is possible to employ a sequence of nucleotides derived from the mouse utrophin gene in a chimaeric construct to provide for expression of a polypeptide with utrophin function.
According to a first aspect of the present invention there is provided a nucleic acid molecule comprising a sequence of nucleotides encoding a polypeptide with utrophin function.
A potypetide with utrophin function is able to bind actin and able to bind the dystrophin protein complex (DPC).
Polypeptides with utrophin function are generally distinguishable immunologically from dystrophin polypeptides. For example, they may comprise at least one epitope not found in dystrophin. Polypeptides with utrophin function may be identified using specific polyclonal or monoclonal antibodies which do not cross-react with dystrophin. If a polypeptide is able to bind actin and able to bind the dystrophin protein complex and at least one antibody can bind it which cannot bind dystrophin, then the polypeptide has utrophin function. In a preferred embodiment, the polypeptide can be bound by an antibody which binds utrophin but not dystrophin, in other words the polypeptide shares at least one epitope with utrophin which epitope is not found in dystrophin. In another embodiment, the polypeptide does not contain an epitope found in dystrophin, such that the polypeptide is not bound by an antibody which binds dystrophin. In such a case, the epitope recognised by the antibody which binds dystrophin may be one not found in utrophin. The polypeptide may contain no epitope found in dystrophin. The immunological comparison may be made with human utrophin and/or dystrophin, especially if the polypeptide with utrophin function is intended for human use, or with the utrophin and/or dystrophin of the species in which use is intended, e.g. mouse. Mouse monoclonal antibodies MANCH07 and MANNUT1 [31] were used in the work described herein. Standard in vitro binding assays may be used to assess immunological cross-reactivity of a polypeptide.
Thus, the polypeptide comprises an actin-binding domain and a dystrophin protein complex (DPC)-binding domain and utrophin-like as opposed to dystrophin-like, e.g. as determined immunologically.
Preferably the encoding sequence comprises a human sequence, i.e. a sequence obtainable from the genome of a human cell.
Comparison of various amino acid sequences reveals the following % similarities (calculated using the method of Needleman and Wunsch (1974) J. Mol. Biol. 48: 443-453, performed using the GAP program from the Winsconsin Package v8, Genetics Computer Group, 575 Science Drive, Madison, Wis. 53711, USA) and identities:
full length human dystrophin v. human utrophin 69% similarity, 50.7% identity;
full length human utrophin v. rat utrophin 93.2% similarity, 87.1% identity;
full length human dystrophin v. mouse dystrophin 95.4% similarity, 91.2% identity;
human dystrophin C-terminus v. human utrophin C-terminus 84.1% similarity, 73.6% identity.
As noted, the present invention is only concerned with xe2x80x9cutrophin-likexe2x80x9d molecules, not xe2x80x9cdystrophin-likexe2x80x9d molecules. Thus, polypeptides according to the present invention (e.g. as encoded by nucleic acid according to the invention) may have an amino acid sequence which is greater than about 75% similar, preferably greater than about 80%, about 85%, about 90%, about 95% or about 98% similarity to the amino acid sequence of FIG. 3 or the amino acid sequence of FIG. 9, taken over the full length. The polypeptides may have an amino acid identity of greater than about 55% identity, preferably greater than about 60% identity, about 70%, about 80%, about 90%, about 95% or about 98% identity over the full-length. The levels of similarity and/or identity may be lower outside the C-terminal, DPC-binding domain provided the DPC-binding domain has greater than about 85% similarity, preferably greater than about 90%, about 95% or about 98% similarity with the DPC-binding domain amino acid sequence of FIG. 3 or FIG. 9, or has greater than about 80%, preferably greater than about 85%, about 90%, about 95% or about 98% identity with the DPC-binding domain amino acid sequence of FIG. 3 or FIG. 9. Particular amino acid sequence variants or derivatives may have a sequence which differs from the sequence of FIG. 3 or FIG. 9 by one or more of insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20, 20-30, 30-50, 50-100, 100-150, or more than 150 amino acids.
The nucleic acid molecule may be an isolate, or in an isolated and/or purified form, that is to say not in an environment in which it is found in nature, removed from its natural environment. It may be free from other nucleic acid obtainable from the same species, e.g. encoding another polypeptide.
The nucleic acid molecule may be one which is not found in nature. For example, the sequence of nucleotides may form part of a cloning vector and/or an expression vector, as discussed further below. The sequence of nucleotides may represent a variant or derivative of a naturally occurring sequence by virtue of comprising an addition, insertion, deletion and/or substitution of one or more nucleotides with respect to the natural sequence, provided preferably that the encoded polypeptide has the specified characteristics. The addition, insertion, deletion and/or substitution of one or more nucleotides may or may not be reflected in an alteration in the encoded amino acid sequence, depending on the genetic code.
Preferably, the nucleic acid molecule is a xe2x80x9cmini-genexe2x80x9d, i.e. the polypeptide encoded does not correspond to full-length utrophin but is rather shorter, a truncated version. For instance, part or all of the rod domain may be missing, such that the polypeptide comprises an actin-binding domain and a DPC-binding domain but is shorter than naturally occurring utrophin. In a full-length utrophin gene, the actin-binding domain is encoded by nucleotides 1-739, while the DPC-binding domain (CRCT) is encoded by nucleotides 8499-10301 (where 1 represents the start of translation; FIG. 2A). The respective domains in the polypeptide encoded by a mini-gene according to the invention may comprise amino acids corresponding to those encoded by these nucleotides in the full-length coding sequence.
Dystrophin mini-genes have been shown to be active in animal models (as discussed). Advantages of a mini-gene over a sequence encoding a full-length utrophin molecule or derivative thereof include easier manipulation and inclusion in vectors, such as adenoviral and retroviral vectors for delivery and expression.
A further preferred non-naturally occurring molecule encoding a polypeptide with the specified characteristics is a chimaeric construct wherein the encoding sequence comprises a sequence obtainable from one mammal, preferably human (xe2x80x9ca human sequencexe2x80x9d), and a sequence obtainable from another mammal, preferably mouse (xe2x80x9ca mouse sequencexe2x80x9d). Such a chimaeric construct may of course comprise the addition, insertion, substitution and/or deletion of one or more nucleotides with respect to the parent mammalian sequences from which it is derived. Preferably, the part of the coding sequence which encodes the actin-binding domain comprises a sequence of nucleotides obtainable from the mouse, or other non-human mammal, or a sequence of nucleotides derived from a sequence obtainable from the mouse, or other non-human mammal.
In a preferred embodiment, the sequence of nucleotides encoding the polypeptide comprises sequence GAGGCAC at residues 332-338 and/or the sequence GATTGTGGATGAAAACAGTGGG at residues 1452-1476 (using the conventional numbering from the initiation codon ATG), and a sequence obtainable from a human.
The nucleic acid molecule may comprise a nucleotide sequence encoding a sequence of amino acids shown in FIG. 1. As discussed, the encoding sequence may be chimaeric, i.e. comprise sequences of nucleotides from different species, e.g. a sequence from or derivable from a human and a sequence from or derivable from a mouse or other non-human mammal.
A chimaeric mini-gene encoding sequence according to the present invention is shown in FIG. 3. Preferred embodiments of the present invention include a nucleic acid molecule comprising a sequence of nucleotides encoding a polypeptide which has an actin-binding domain and a DPC-binding domain and which polypeptide comprises an amino acid sequence encoded by a sequence of nucleotides shown in FIG. 3, a nucleic acid molecule comprising a sequence of nucleotides encoding a variant, allele. or derivative of such a polypeptide by way of addition, substitution, insertion and/or deletion of one or more amino acids, and a nucleic acid molecule comprising a sequence of nucleotides which is a variant, allele or derivative of the sequence shown in FIG. 3, by way of addition, substitution, insertion and/or deletion of one or more nucleotides, with or without a change in the encoded amino acid sequence with respect to the amino acid sequence encoded by a sequence of nucleotides shown in FIG. 3. The proviso is that the encoded polypeptide is xe2x80x9cutrophin-likexe2x80x9d rather than xe2x80x9cdystrophin-likexe2x80x9d, e.g. as determined immunologically as discussed.
One particular variant or derivative of the sequence of FIG. 3 has a sequence as shown in FIG. 9, which is a xe2x80x9cfull-lengthxe2x80x9d utrophin construct, including rod domain sequences not included in the mini-gene of FIG. 3.
The sequences of FIG. 3 and FIG. 9 include some positions at which the precise residue is left open (marked by xe2x80x9cNxe2x80x9d in the nucleotide sequence and xe2x80x9cXxe2x80x9d in the amino acid sequence). Comparison of the human, mouse and rat utrophin sequences in this region (FIG. 10) shows that the human and rat amino acid sequences are absolutely conserved here. Accordingly, the twelve xe2x80x9cX""sxe2x80x9d in FIGS. 3 and 9 may represent the amino acid sequence DKKSIIMYLTSL (SEQ ID NO:15). Instead, in accordance with the discussion of variants and derivatives herein, a polypeptide according to the invention (as encoded by nucleic acid according to the invention) may include a variant or derivative sequence, by way of one or more of insertion, addition, substitution or deletion of one or more amino acids of the sequence DKKSIIMYLTSL (SEQ ID NO:15), in the position marked by the X""s in FIGS. 3 and 9.
Nucleic acid according to the present invention is obtainable by hybridising nucleic acid of target cells (e.g. human, mouse, rat) with one or more oligo- or poly-nucleotides with sequences designed based on the sequence information presented in FIG. 1, FIG. 3 or FIG. 9. Thus, the full mouse sequence, or the sequence in the region marked by the X""s in FIGS. 3 and 9, may be obtained by probing or PCR using sequence information provided herein (e.g. FIG. 1).
Nucleic acid according to the present invention is obtainable using one or more oligonucleotide probes or primers designed to hybridise with one or more fragments of a nucleic acid sequence shown in FIG. 1, FIG. 3 or FIG. 9, particularly fragments of relatively rare sequence, based on codon usage or statistical analysis. The amino acid sequence information provided may be used in design of degenerate probes/primers or xe2x80x9clongxe2x80x9d probes. A primer designed to hybridise with a fragment of the nucleic acid sequence shown may be used in conjunction with one or more oligonucleotides designed to hybridise to a sequence in a cloning vector within which target nucleic acid has been cloned, or in so-called xe2x80x9cRACExe2x80x9d (rapid amplification of cDNA ends) in which cDNA""s in a library are ligated to an oligonucleotide linker and PCR is performed using a primer which hybridises with the sequence shown in the figure and a primer which hybridises to the oligonucleotide linker.
Nucleic acid isolated and/or purified from one or more cells (e.g. human, mouse) or a nucleic acid library derived from nucleic acid isolated and/or purified from cells (e.g. a cDNA library derived from mRNA isolated from the cells), may be probed under conditions for selective hybridisation and/or subjected to a specific nucleic acid amplification reaction such as the polymerase chain reaction (PCR).
A method may include hybridisation of one or more (e.g. two) probes or primers to target nucleic acid. Where the nucleic acid is double-stranded DNA, hybridisation will generally be preceded by denaturation to produce single-stranded DNA. The hybridisation may be as part of a PCR procedure, or as part of a probing procedure not involving PCR. An example procedure would be a combination of PCR and low stringency hybridisation. A screening procedure, chosen from the many available to those skilled in the art, is used to identify successful hybridisation events and isolated hybridised nucleic acid.
Probing may employ the standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes. Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined. DNA for probing may be prepared from RNA preparations from cells.
Preliminary experiments may be performed by hybridising under low stringency conditions various probes to Southern blots of DNA digested with restriction enzymes. Suitable conditions would be achieved when a large number of hybridising fragments were obtained while the background hybridisation was low. Using these conditions nucleic acid libraries, e.g. cDNA libraries representative of expressed sequences, may be searched.
It may be necessary for one or more gene fragments to be ligated to generate a full-length coding sequence. Also, where a full-length encoding nucleic acid molecule has not been obtained, a smaller molecule representing part of the full molecule, may be used to obtain full-length clones. Inserts may be prepared from partial cDNA clones and used to screen cDNA libraries.
Those skilled in the art are well able to employ suitable conditions of the desired stringency for selective hybridisation, taking into account factors such as oligonucleotide length and base composition, temperature and so on.
Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells and many others. A common, preferred bacterial host is E. coli. 
Nucleic acid according to the present invention may form part of a cloning vector and/or a vector from which the encoded polypeptide may be expressed. Suitable vectors can be chosen or constructed, containing appropriate and appropriately positioned regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2 nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley and Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. are incorporated herein by reference.
Thus, a further aspect of the present invention provides a host cell containing nucleic acid as disclosed herein. A still further aspect provides a method comprising introducing such nucleic acid into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.
The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene.
In one embodiment, the nucleic acid of the invention is integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.
The invention also provides a mammal, such as a human, primate or rodent, preferably rat or mouse, comprising a host cell as provided, and methods of production and use of such a mammal. The mammal may be non-human. Transgenic animals, particularly mice, can be generated using any available technique. Particularly suitable for purposes of study are mdx mice or others with a dystrophic phenotype.
The polypeptide encoded by the nucleic acid may be expressed from the nucleic acid in vitro, e.g. in a cell-free system or in cultured cells, or in vivo. In vitro expression may be useful in determining ability of the polypeptide to bind to actin and/or DPC. This may be useful in testing or screening for substances able to modulate one or both of these binding activities. In particular, substances able to increase actin and/or DPC binding of the polypeptide will add to the repertoire of molecules available for potential pharmaceutical/therapeutic exploitation. Such substances, identified as modulators of one or both of the binding activities of the polypeptide, following expression of the polypeptide from encoding nucleic acid therefor, may be investigated further and may be manufactured and/or used in preparation of a medicament, pharmaceutical composition or drug which may subsequently be administered to an individual. In vivo expression is discussed further below.
According to a further aspect of the present invention there is provided a polypeptide with utrophin function (other than utrophin itself). Such a polypeptide comprises an actin-binding domain and a DPC-binding domain and is immunologically recognisable as utrophin-like rather than dystrophin-like, as discussed, not-being a naturally occurring polypeptide. The polypeptide may be any of those discussed above as being encoded by nucleic acid according to the present invention. In particular, the polypeptide may be shorter than naturally occurring full-length utrophin, for example by virtue of lacking all or part of the rod domain. The actin-binding and DPC-binding domains may correspond to those of human, mouse or other non-human utrophin or may be derived therefrom by way of addition, substitution, insertion and/or deletion of one or more amino acids. The polypeptide may be chimaeric, comprising sequences of amino acids from or derived from different species, e.g. human and mouse, as discussed.
A convenient way of producing a polypeptide according to the present invention is to express nucleic acid encoding it. Accordingly, methods of making such polypeptides by expression from encoding nucleic acid therefor are provided by the present invention, in vitro, e.g. in cell-free systems or by culturing host cells under appropriate conditions for expression, or in vivo.
Polypeptides and nucleic acid according to the invention may be used in the manufacture of medicaments, compositions, including pharmaceutical formulations, and drugs for delivery to an-individual, e.g. a human with muscular dystrophy or a non-human mammal, such as a mouse, as a model for study of the polypeptides, muscular dystrophy and therapy thereof.
For example, a method of treatment practised on the human or animal body in accordance with the present invention may comprise administration to an individual of nucleic acid encoding a polypeptide as disclosed herein. The nucleic acid may form part of a construct enabling expression within cells of the individual. Nucleic acid may be introduced into cells using a retroviral vector, preferably one which will not transform cells, or using liposome technology.
Administration is preferably in a xe2x80x9ctherapeutically effective amountxe2x80x9d, this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, eg decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors.
A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.
Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such has water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer""s Injection, Lactated Ringer""s Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
Injection may be used to deliver nucleic acid to disease sites. Internally, suitable imaging devices may be employed to guide an injecting needle to the desired site.
It may be desirable to remove cells from the body, treat them then return them to the body, or to administer cells derived from cells removed from an individual. This might be appropriate, for example, if muscle stem cells can be isolated. Muscle precursor cells (xe2x80x9cmpcxe2x80x9d) have been used in cell therapy in mdx mice, where implantation of normal mpc gave rise to substantial amounts of dystrophin [25,26,27]. Immunosuppression increases success of cell implantation procedures [13]. Myoblasts may be used to introduce genes into muscle fibres during growth or repair, as has been demonstrated using a replication-defective retroviral vector to introduce a mini-dystrophin construct into proliferating myogenic cells in tissue culture [28].
Thus, cells in culture may have nucleic acid according to the present invention introduced into them before the cells are grafted into muscles in a patient. Grafting the cells back into the donor has the advantages of a genetically corrected autologous transplant. Nucleic acid may be introduced locally into cells using transfection, electroporation, microinjection, liposomes, lipofecting or as naked DNA or RNA, or using any other suitable technique.
Retroviral vectors have also been used to introduce the dystrophin mini-gene into the myoblasts of spontaneously regenerating muscle of the mdx mouse to produce dystrophin-positive fibres [8]. Recombinant replication defective adenoviruses appear particularly effective as an efficient means of introducing constructs into skeletal muscle fibres for persistent expression [29]. See reference 11 for a review of myoblast-based gene therapies.
Adenoviral, retroviral or other viral vectors may be used advantageously for the introduction of a utrophin sequence according to the present invention into muscle cells. Even though in vivo transduction may be restricted to growing or regenerating muscle fibres, retrovirally introduced constructs have the advantage of becoming integrated into the genome of the host cell, potentially conferring lifelong expression.
Liposomes may be used as vehicles for delivery of nucleic acid constructs to skeletal muscle. Intravenous injection of constructs in cationic liposomes has resulted in widespread transfection of most tissues, including skeletal muscle [30]. Lack of immunogenicity allows for repeated administration and lack of tissue specificity may be accommodated by choosing a muscle-specific promoter to drive expression.
For use in distinguishing polypeptide with utrophin function from dystrophin and related polypeptides, antibodies may be obtained using techniques which are standard in the art. Methods of producing antibodies include immunising a mammal (eg mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof, or a cell or virus which expresses the protein or fragment. Immunisation with DNA encoding a target polypeptide is also possible (see for example Wolff, et al. Science 247: 1465-1468 (1990); Tang, et al. Nature 356: 152-154 (1992); Ulmer J B, et al. Science 259: 1745-1749 (1993)). Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al, 1992, Nature 357: 80-82).
The production of monoclonal antibodies is well established in the art. Monoclonal antibodies can be subjected to the techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP184187A, GB 2188638A or EP-A-0239400. A hybridoma producing a monoclonal antibody may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.
As an alternative or supplement to immunising a mammal with a peptide, an antibody specific for a protein may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, eg using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces;. for instance see WO92/01047. The library may be naive, that is constructed from sequences obtained from an organism which has not been immunised with the target, or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest (or a fragment thereof).
Antibodies may be modified in a number of ways. Indeed the term xe2x80x9cantibodyxe2x80x9d should be construed as covering any specific binding substance having an binding domain with the required specificity. Thus this covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or synthetic. Chimaeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimaeric antibodies are described in EP-A-0120694 and EP-A-0125023.
It has been shown that the function of binding antigens can be performed by fragments of a whole antibody. Example binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E. S. et al., Nature 341, 544-546 (1989)) which consists of a VH domain; (v) isolated CDR regions; (vi) F(abxe2x80x2)2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) xe2x80x9cdiabodiesxe2x80x9d, multivalent or multispecific fragments constructed by gene fusion (WO94/13804; P. Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993).
Further aspects and embodiments of the present invention, and modifications to aspects and embodiments disclosed herein, will be apparent to those skilled in the art.